The long term objective of this research is to identify the biochemical mechanisms by which protein serine/threonine kinases are regulated in cells stimulated with growth factors. Growth factors regulate a variety of cellular processes, including growth, differentiation, embryogenesis and wound repair. Aberrant responses of cells to growth factors are strongly implicated in many diseases, such as cancer, diabetes and birth defects. When cells are treated with growth factors, a large number of proteins undergo changes in their state of phosphorylation. Thus, phosphorylation is a major mechanism for rapidly altering the molecular properties of protein targets. Although growth factor signalling often begins with activation of receptor tyrosine kinases, the vast majority of intracellular targets for phosphorylation are modified on serine or threonine residues. Therefore, identifying the pathways by which receptor tyrosine kinases communicate with protein serine/threonine kinases is key to understanding how cells respond to external stimuli. A specific pathway has been found by which growth factors regulate phosphorylation through the stimulation of serine/threonine kinases. Three enzymes in this pathway have been identified, named pp90 ribosomal S6 kinase (pp90rsk), ERK (extracellular signal regulated kinase), and ERK kinase. All of these enzymes catalyze phosphorylation reactions and in addition are regulated by phosphorylation; together they form three successive tiers of a protein kinase cascade in which ERK kinase phosphorylates and activates ERK, and ERK phosphorylates and activates pp90rsk. The factors that directly regulate ERK kinase, and thus function as intermediates between this enzyme and receptor tyrosine kinases, are so far unidentified. The specific aims of this project are to (i) investigate the mechanism by which ERK kinase activates ERK, (ii) identify a proposed kinase acting upstream of ERK kinase and downstream of growth factor receptor tyrosine kinases, which activates ERK kinase by serine phosphorylation, (iii) confirm preliminary indications that the kinase cascade is reversibly deactivated by protein phosphatases and examine how deactivation is regulated, and (iv) test whether the pathway works in intact cells as it does in vitro. In the work proposed, cultured mammalian cell systems will be studied using biochemical,pharmacological, and molecular biological approaches to examine the regulation of the kinase cascade in vitro and in intact cells. The information gained from these studies will lay the groundwork for understanding the factors that control this important signal transduction pathway at a molecular and mechanistic level.